The retroviral CA (or capsid) protein has several essential roles in the virus life cycle organizing the packing of the Gag polyprotein during immature particle assembly, directing morphogenesis of the mature capsid in the virion core, and finally, acting in poorly defined role(s) during the early stages of replication. The ability of the CA protein to undergo complex molecular rearrangements and conformational changes is critical for its ability to support the different functions in the viral life cycle. The carboxy-terminal domain (CTD) of the protein is a structural module that participates in intermolecular interactions between Gag polyproteins in the immature particle and in CA-CA association after the maturation process cleaves Gag, rearranges the constituents and activates the virion core for subsequent reverse transcription. A role for highly conserved residues in the CTD in contributing to the intermolecular interactions both before and after maturation has been indicated by genetic analyses and structural studies involving several retroviruses. The work of the Craven laboratory in analyzing a large set of mutations in the Rous sarcoma virus CA protein has documented the dual role of this major homology region in both immature and mature virion function and demonstrated the ability of secondary substitutions at distant and diverse sites to compensate for lethal mutations in this area. In particular, a hot spot for suppression of these mutations has been mapped to the second 1-helix of the CTD. The findings suggest that this region of the protein works together with the conserved motif to control a critical conformational switch needed for correct maturation of the capsid. The proposed experiments will combine the genetic and biophysical expertise of two investigators to directly test the role of the 12 helix of the CTD in achieving a protein conformation that is competent for assembly of functional capsids. Specifically, these studies will use NMR spectroscopy to assess changes in the three-dimensional structure of the CTD upon introduction of particular lethal and suppressor mutations. In addition a series of different amino acids will be substituted at one position in the CTD which has been shown to be a particular hot spot for suppression of lethal MHR mutations. The structural effects caused by different substitutions of the CTD will be correlated with their in vivo effects on virus replication, morphology, and core stability. It is expected that this work will enable us define the contribution of the 12 helix to a conformational switch that is critical for retrovirus maturation and which is likely to have similar counterparts in other viral systems. PUBLIC HEALTH RELEVANCE: In retroviruses such as HIV, the human T-cell lymphotropic virus and the Rous sarcoma virus, a stage of the virus life cycle known as maturation involves very dramatic structural changes in the interior of the virus particle, leading to the activation of its infectious potential. The studies described in this proposal will use a combination of genetic and protein structural approaches to examine the molecular mechanisms that control this process. A detailed understanding of this essential step of virus infection will allow development of better maturation inhibitors for use as antiretroviral drugs.